Autoreceptor-induced inhibition of neuropeptide Y release from PC-12 cells is mediated by Y2 receptors.

Pheochromocytoma (PC)-12 cells express Y1, Y2, and Y3 neuropeptide Y (NPY) receptors when differentiated with nerve growth factor (NGF). The present work evaluated NGF-differentiated PC-12 cells as a model system to study modulation of NPY release by NPY autoreceptors. We demonstrated that both K+ and nicotine stimulated concomitant release of NPY and dopamine from differentiated PC-12 cells. We also showed in this study that NPY release from PC-12 cells was attenuated in a concentration-dependent manner by peptide YY (PYY)-(13-36), a selective agonist for the Y2 type of NPY receptors. This result demonstrated that NPY release could be modulated by NPY autoreceptors of the Y2 subtype. The inhibitory action of PYY-(13-36) may be mediated at least in part by inhibition of N-type Ca2+ channels, because PYY-(13-36) could not produce further inhibitory effects in the presence of a maximum effective concentration of omega-conotoxin, an N-type Ca2+-channel blocker. The inhibition by PYY-(13-36) could be blocked by pretreatment of cells with pertussis toxin, suggesting that an inhibitory GTP-binding protein was involved. Furthermore, the function of NPY autoreceptors could be modulated by other receptors such as beta-adrenergic and ATP receptors. The evoked release of NPY was also attenuated by ATP and adenosine, which have been shown to be colocalized and coreleased with NPY from sympathetic nerve terminals. These results suggest that PC-12 cells differentiated with NGF may be an ideal model to study regulatory mechanisms of NPY release and that autoreceptor-mediated regulation of NPY release appears to act through the Y2 subtype of the NPY receptor.

Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor.

To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).

Alterations in neurokinin 1 receptor gene expression in models of pain and inflammation.

Substance P and the related tachykinin peptides are involved in inflammatory processes and in the transmission of sensory nociceptive information. In this article we review the evidence implicating substance P and the neurokinin 1 (NK1) receptor in arthritic disease. We also provide preliminary evidence demonstrating that cultured synoviocytes from a patient with rheumatoid arthritis express NK1 receptor mRNA that can be downregulated by tumor necrosis factor alpha, whereas synoviocytes from a normal patient do not express detectable NK1 receptor mRNA or protein. Data are also presented summarizing recent studies on nociception-induced increases in sensory ganglia of levels of mRNA encoding substance P and increases in dorsal horn NK1 receptor mRNA levels. Morphine pretreatment blocked the increases in dorsal horn NK1 receptor mRNA levels but did not block the nociception-induced substance P encoding mRNA levels in sensory ganglia. These results are discussed with reference to mechanisms that may regulate P turnover and NK1 receptor sensitivity in models of pain and inflammation.

Effects of differentiation on neuropeptide-Y receptors and responses in rat pheochromocytoma cells.

Undifferentiated rat pheochromocytoma PC12 cells resemble immature adrenal chromaffin cells, express neuropeptide-Y (NPY) receptors of the Y1 subtype, and synthesize catecholamines as well as NPY. In the present study, we examined how phenotypic alteration of PC12 cells by nerve growth factor (NGF) or glucocorticoid affected cellular responsiveness to NPY and related agonists, especially with regard to modulation of catecholamine overflow. Unlike undifferentiated PC12 cells, cells differentiated to a sympathetic neuronal phenotype with NGF were responsive to the Y2 receptor-selective agonist, NPY 13-36. NPY 13-36 1) inhibited binding of [125I]NPY 1-36, 2) inhibited accumulation of evoked cAMP, and 3) inhibited evoked catecholamine overflow. NGF-differentiated cells were also responsive to the Y1 receptor-selective agonist [Leu31,Pro34]NPY (LP-NPY). Like NPY-(13-36), LP-NPY inhibited binding of [125I]NPY-(1-36); however, LP-NPY and NPY-(13-36) exerted their effects through heterogeneous receptors, as LP-NPY enhanced while NPY 13-36 inhibited evoked catecholamine overflow in NGF-differentiated cells, despite the fact that both agonists inhibited the evoked cAMP. In contrast to NGF-differentiated cells, cells differentiated to a mature chromaffin phenotype with dexamethasone were unresponsive to NPY-(13-36), nor did the Y2 agonist inhibit binding of [125I]NPY-(1-36). Dexamethasone-differentiated PC12 cells were, however, responsive to LP-NPY, as this agonist enhanced evoked catecholamine overflow and inhibited binding of [125I]NPY-(1-36). Peptide-YY also enhanced catecholamine overflow, but only significantly at 100 nM. The data suggest differential expression of NPY receptor subtypes on neuronal and endocrine cells where catecholamine overflow is a key feature. These studies further demonstrate inhibitory or excitatory modulation of catecholamine transmission by NPY via distinct receptor subtypes in homogeneous sympathoadrenomedullary models resembling sympathetic neurons and chromaffin cells.

Diminished neuropeptide Y and dopamine beta-hydroxylase immunoreactivity in a guinea pig model of left ventricular hypertrophy.

OBJECTIVE: The aims of the study were to determine the effect of chronic pressure overload of the left ventricle on the density and distribution of neuropeptide-Y-like immunoreactive (NPY-LI) nerve fibres in heart and to compare any changes to those observed in adrenergic nerve fibres, identified by dopamine beta-hydroxylase immunoreactivity. METHODS: Pressure overload was produced in female adult guinea pigs by constriction of the abdominal aorta, using a modified Weck haemoclip. The same operation was performed on a separate group of animals except that no clip was placed around the aorta. Five weeks after surgery, animals were anaesthetised, and the hearts were fixed by perfusion for immunohistochemistry. Cryostat sections were stained, using an indirect peroxidase/antiperoxidase method, for NPY or dopamine beta-hydroxylase. RESULTS: Aortic stenosis caused a 45% increase in left ventricular weight and a 58% increase in left atrial weight at 5 weeks postsurgery. Pulmonary oedema, a sign of cardiac failure, was evident in most of the animals with aortic stenosis. Immunohistochemical studies showed that in atria and right ventricles from animals with abdominal aortic stenosis the distribution and density of NPY-LI nerve fibres were similar to those in the sham operated guinea pigs. However, the left ventricles obtained from the animals with aortic stenosis were nearly devoid of NPY-LI nerve fibres. The density of dopamine beta-hydroxylase-LI nerve fibres was also substantially reduced in the hypertrophied left ventricles. CONCLUSIONS: Aortic stenosis resulting in left ventricular hypertrophy caused a nearly complete loss of NPY-LI and dopamine beta-hydroxylase-LI nerve fibres from the left ventricle. The parallel reduction in both neuropeptide Y and dopamine beta-hydroxylase is in accordance with the association of neuropeptide Y with sympathetic (adrenergic) nerve fibres in the left ventricle and suggests that chronic left ventricular hypertrophy causes a severe degeneration of sympathetic axons supplying this chamber and/or reduces the ability of these sympathetic neurones to maintain normal levels of neurotransmitter related enzymes and neuropeptides.

An endogenous ligand for the sigma opioid binding site.

It had been suggested that phencyclidine (PCP) and sigma opioids exert their similar psychotomimetic effects through a common receptor. Recently, however, there have been several reports demonstrating significant differences between the binding of PCP and SKF 10,047, a sigma opioid agonist, which suggests that there may be distinct PCP and sigma opioid receptors. If these differences in binding represent different receptors, then there may be different endogenous ligands for each receptor. Using porcine brains, which have already been used to isolate and purify an endogenous ligand for the PCP receptor, another factor has been isolated that inhibited the binding of [3H]-(+)SKF 10,047 and not the binding of [3H]-PCP. This factor appears to be a peptide or protein because incubation of the active fraction with pronase, a nonspecific peptidase, eliminated the ability of the porcine fractions to inhibit the binding of [3H]-(+)SKF 10,047. These findings suggest the existence of an endogenous ligand for sigma opioid receptors, which is different from the previously identified endogenous ligand for PCP receptors.

Phencyclidine. Physiological actions, interactions with excitatory amino acids and endogenous ligands.

Phenycyclidine (PCP) produces many profound effects in the central nervous system. PCP has numerous behavioral and neurochemical effects such as inhibiting the uptake and facilitating the release of dopamine, serotonin, and norepinephrine. PCP also interacts with sigma, mu opioid, muscarinic, and nicotinic receptors. However, the psychotomimetic effects induced by PCP are believed to be mediated by specific PCP receptors, where PCP binds with greater potency than sigma compounds. Electrophysiological, behavioral, and neuro-chemical evidence strongly suggests that at least some of the many PCP actions result from antagonism of excitatory amino acid-induced responses via PCP receptors. The recent isolation and partial characterization of the alpha and beta endopsychosins and the identification of other endogenous ligands for the PCP and sigma receptors, is another promising area of research in the elucidation of the physiological role of an endogenous PCP and sigma system.

Enkephalin and neuropeptide Y: two colocalized neuropeptides are independently regulated in primary cultures of bovine chromaffin cells.

We have found that Neuropeptide Y is colocalized with enkephalin in bovine adrenal chromaffin cells. The two peptides can be found in the same granules in those cells where they coexist. These cells correspond to the adrenergic subpopulation of chromaffin cells since they contain the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase. Despite their coexistence, production of the two peptides is independently regulated. Enkephalin levels are doubled after nicotinic depolarization (which increases enkephalin synthesis) or after treatment with reserpine (which increases enkephalin precursor processing). Neither of these treatments, acting by different mechanisms, has any effect on the levels of Neuropeptide Y.

Neuropeptide Y immunoreactivity in human cerebrospinal fluid.

Neuropeptide Y (NPY) immunoreactivity has been determined in human cerebrospinal fluid (CSF). High pressure liquid chromatography revealed that the NPY immunoreactivity co-eluted with authentic NPY. The range of NPY levels was 108 +/- 18 pg/ml (mean +/- S.D.) in a group of 28 normal subjects. In five additional subjects NPY immunoreactivity was measured in 4 sequential 8 ml aliquots of CSF to determine whether a rostro-caudal gradient was present. No significant differences in NPY levels were detected between any of the 4 aliquots.

Amphetamine-induced changes in immunoreactive NPY in rat brain, pineal gland and plasma.

Acute injection of d-amphetamine (10 mg/kg), administered to rats 60 minutes prior to sacrifice, induced a doubling of immunoreactive NPY (NPY-IR) in pineal gland. No changes, however, could be detected in levels of NPY-IR in grossly dissected or microdissected regions of rat brain, nor were changes evident in plasma level concentrations of NPY-IR following acute amphetamine pretreatment. When amphetamine was injected twice daily for six days and once more 60 minutes prior to sacrifice, levels of NPY-IR were decreased in caudate putamen and the paraventricular and dorsomedial nuclei of the hypothalamus, while concentrations of NPY-IR were increased in medial preoptic nucleus, pineal gland, and plasma. These data indicate that levels of NPY-IR are susceptible to manipulation by amphetamine, where the extent and direction of change (increase or decrease) depends on both the frequency of drug administration and the nature of the sampled tissue. Based on the effects of amphetamine on central and peripheral norepinephrine and epinephrine disposition observed in other studies, the data also suggest that NPY-IR and catecholamine dispositions are not directly correlated and may be inversely related in some tissue.

Pancreatic polypeptide immunoreactivity in rat brain is actually neuropeptide Y.

Radioimmunoassay was combined with high pressure liquid chromatography and immunohistochemistry to establish the identity of pancreatic polypeptide-like immunoreactive material in the central nervous system of the rat. Antisera to avian pancreatic polypeptide, bovine pancreatic polypeptide, the invariant amidated carboxyterminal hexapeptide fragment of mammalian pancreatic polypeptides and the structurally related peptide, neuropeptide Y, were used immunocytochemically to localize neurons containing immunoreactive pancreatic polypeptide-like material in rat brain. Adjacent brain sections stained by the indirect immunofluorescent technique and single sections from double-staining experiments demonstrated that identical fibers and perikarya stained for pancreatic polypeptide-like immunoreactive material by antisera directed against each of the four peptides. Characterization of pancreatic polypeptide-like immunoreactive material in chromatographed rat brain extracts by radioimmunoassay using antisera to either neuropeptide Y or the carboxy-terminal portion of the pancreatic polypeptide molecule revealed that the major peak of immunoreactive material, as measured by either assay, appeared to co-elute with synthetic porcine neuropeptide Y. A minor peak of immunoreactive material co-eluting with peptide YY standard was indicated by the neuropeptide Y radioimmunoassay. This was contrasted by data obtained from chromatographic profiles of rat pancreas, which showed that the main immunoreactive peak, as measured by the neuropeptide Y assay, co-eluted with porcine peptide YY, with a minor peak co-eluting with porcine neuropeptide Y. The main peak of immunoreactive material in pancreas, as measured by the pancreatic polypeptide carboxy-terminal radioimmunoassay, eluted considerably earlier than standard peptide YY, neuropeptide Y and bovine pancreatic polypeptide, and is probably identical to rat pancreatic polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The anatomy of neuropeptide-Y-containing neurons in rat brain.

The distribution of neuropeptide Y in the central nervous system of adult male rats was investigated using indirect immunofluorescence, the peroxidase-antiperoxidase technique and by radioimmunoassay of microdissected brain regions. The different methods were in good agreement and showed that neuropeptide Y had a widespread distribution and was present in extremely high concentrations. The highest concentrations of neuropeptide Y were found in the paraventricular hypothalamic nucleus and hypothalamic arcuate nucleus, which also contained the highest density of immunoreactive fibers and numbers of perikarya, respectively. The suprachiasmatic nucleus, median eminence, dorsomedial hypothalamic nucleus and paraventricular thalamic nucleus showed high concentrations as well as high densities of fibers. Moderate concentrations were found in the bed nucleus of the stria terminalis, although a high density of fibers was found. Areas with moderate concentrations and densities of fibers were the medial preoptic area, anterior hypothalamic area, periventricular nucleus, posterior hypothalamus and the medial amygdaloid nucleus. The nucleus of the solitary tract contained a low concentration of neuropeptide Y although a high number of immunoreactive perikarya was found in colchicine-treated rats. Low concentrations were also measured in the cerebral cortex, yet relatively high numbers of cell bodies and fibers were found dispersed through the cortex. The extremely high concentrations and widespread distribution of neuropeptide Y in the central nervous system suggests a number of important physiological roles for this neurotransmitter candidate.

Neuropeptide Y and peptide YY neuronal and endocrine systems.

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.

Evidence for an endogenous peptide ligand and antagonist for PCP receptors.

alpha-Endopsychosin, an endogenous ligand for the phencyclidine receptor, has been isolated from porcine brain. The endogenous ligand is a peptide and has similar actions to PCP and is selectively distributed in the brain. A PCP analogue, Metaphit, has also been found to be an effective PCP antagonist. This antagonist may be useful for evaluating the role of the brain's alpha-endopsychosin system and may have a number of important clinical uses.

Evidence for an endogenous peptide ligand for the phencyclidine receptor.

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.